This invention is in the field of medicinal chemistry and relates to novel compounds, and pharmaceutical compositions thereof, that inhibit caspases that mediate cell apoptosis and inflammation. The invention also relates to methods of using the compounds and pharmaceutical compositions of this invention to treat diseases where caspase activity is implicated.
Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders (see generally Science, 1998, 281, 1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).
Caspases are a family of cysteine protease enzymes that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathways vary depending on cell type and stimulus, but all apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.
Caspase-1, the first identified caspase, is also known as interleukin converting enzyme or xe2x80x9cICE.xe2x80x9d Caspase-1 converts precursor interleukin-1xcex2 (xe2x80x9cpIL-1xcex2xe2x80x9d) to the pro-inflammatory active form by specific cleavage of pIL-1xcex2 between Asp-116 and Ala-117. Besides caspase-1 there are also eleven other known human caspases, all of which cleave specifically at aspartyl residues. They are also observed to have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The group of caspases, which includes caspases 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. Another group which includes caspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10, tolerate many amino acids in the primary recognition sequence, but seem to prefer residues with branched, aliphatic side chains such as valine and leucine at position 4.
The caspases have also been grouped according to their perceived function. The first subfamily consists of caspases-1 (ICE), 4, and 5. These caspases have been shown to be involved in pro-inflammatory cytokine processing and therefore play an important role in inflammation. Caspase-1, the most studied enzyme of this class, activates the IL-1xcex2 precursor by proteolytic cleavage. This enzyme therefore plays a key role in the inflammatory response. Caspase-1 is also involved in the processing of interferon gamma inducing factor (IGIF or IL-18) which stimulates the production of interferon gamma, a key immunoregulator that modulates antigen presentation, T-cell activation and cell adhesion.
The remaining caspases make up the second and third subfamilies. These enzymes are of central importance in the intracellular signaling pathways leading to apoptosis. One subfamily consists of the enzymes involved in initiating events in the apoptotic pathway, including transduction of signals from the plasma membrane. Members of this subfamily include caspases-2, 8, 9 and 10. The other subfamily, consisting of the effector capsases 3, 6 and 7, are involved in the final downstream cleavage events that result in the systematic breakdown and death of the cell by apoptosis. Caspases involved in the upstream signal transduction activate the downstream caspases, which then disable DNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton and finally fragment the cell.
A four amino acid sequence primarily recognized by the caspases has been determined for enzyme substrates. Talanian et al., J. Biol. Chem. 272, 9677-9682, (1997); Thornberry et al., J. Biol. Chem. 272, 17907-17911, (1997). Knowledge of the four amino acid sequence primarily recognized by the caspases has been used to design caspase inhibitors. Reversible tetrapeptide inhibitors have been prepared having the structure CH3COxe2x80x94[P4]xe2x80x94[P3]xe2x80x94[P2]xe2x80x94CH(R)CH2CO2H where P2 to P4 represent an optimal amino acid recognition sequence and R is an aldehyde, nitrile or ketone capable of binding to the caspase cysteine sulfhydryl. Rano and Thornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med. Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43 (1995). Irreversible inhibitors based on the analogous tetrapeptide recognition sequence have been prepared where R is an acyloxymethylketone-COCH2OCORxe2x80x2. Rxe2x80x2 is exemplified by an optionally substituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH2X where X is a leaving group such as F or Cl. Thornberry et al., Biochemistry 33, 3934 (1994); Dolle et al., J. Med. Chem. 37, 563-564 (1994).
The utility of caspase inhibitors to treat a variety of mammalian disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. For example, in rodent models, caspase inhibitors have been shown to reduce infarct size and inhibit cardiomyocyte apoptosis after myocardial infarction, to reduce lesion volume and neurological deficit resulting from stroke, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury, to be effective in treating fulminant liver destruction, and to improve survival after endotoxic shock. Yaoita et al., Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585 (1999).
In general, the peptidic inhibitors described above are very potent against some of the caspase enzymes. However, this potency has not always been reflected in cellular models of apoptosis. In addition peptide inhibitors are typically characterized by undesirable pharmacological properties such as poor oral absorption, poor stability and rapid metabolism. Plattner and Norbeck, in Drug Discovery Technologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England, 1990).
There are reports of modified peptide inhibitors. WO 91/15577 and WO 93/05071 disclose peptide ICE inhibitors of the formula:
Z-Q2-Asp-Q1 
wherein Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids; and Q1, is an electronegative leaving group.
WO 99/18781 discloses dipeptide caspase inhibitors of the formula: 
wherein R1 is an N-terminal protecting group; AA is a residue of a natural xcex1-amino acid or xcex2-amino acid; R2 is hydrogen or CH2R4 where R4 is an electronegative leaving group; and R3 is alkyl or hydrogen.
WO 99/47154 discloses dipeptide caspase inhibitors of the formula: 
wherein R1 is an N-terminal protecting group; AA is a residue of a non-natural xcex1-amino acid or xcex2-amino acid; and R2 is optionally substituted alkyl or hydrogen.
WO 00/023421 discloses (substituted) acyl dipeptide apoptosis inhibitors having the formula: 
where n is 0, 1, or 2; q is 1 or 2; A is a residue of certain natural or non-natural amino acid; B is a hydrogen atom, a deuterium atom, C1-10 straight chain or branched alkyl, cycloalkyl, phenyl, substituted phentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH2)mcycloalkyl, (CH2)mphenyl, (CH2)m(substituted phenyl), (CH2)m(1- or 2-naphthyl), (CH2)mheteroaryl, halomethyl, CO2R13, CONR14R15, CH2ZR16, CH2OCOaryl, CH2OCO (substituted aryl), CH2OCO (heteroaryl), CH2OCO (substituted heteroaryl), or CH2OPO (R17) R18, where R13, R14,R15, R16, R17 and R18 are defined in the application; R2 is selected from a group containing hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, (CH2)mNH2; R3 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is CH2, C=O, O, S, NH, C=ONH or CH2OCONH; and Z is an oxygen or a sulfur atom.
WO 97/24339 discloses inhibitors of interleukin-1xcex2 converter enzyme of the formula: 
wherein R1 represents H, alkyl, alkoxy, a carbocycle, a heterocycle, and various other groups; AA1 and AA2 are single bonds or amino acids; and Y represents a group of formula: 
wherein the Tet ring represents a tetrazole ring; and Z represents, inter alia, alkylene, alkenylene, O, S, SO, and SO2.
EP 618223 discloses ICE inhibitors of the formula:
Rxe2x80x94A1xe2x80x94A2xe2x80x94Xxe2x80x94A3 
wherein R is H, a protecting group, or an optionally ring substituted PhCH2O; A1 is an xcex1-hydroxy- or xcex1-amino acid residue; A2 is an xcex1-hydroxyacid residue or xcex1-amino acid or A1 and A2 form together a pseudodipeptide or a dipeptide mimetic residue; X is a residue derived from Asp wherein A3 is CH2X1COY1, CH2OY2, CH2SY3 or CH2(CO)mY6 wherein X1 is O or S, m is 0 or 1 and Y1, Y2, Y3 and Y6 are optionally substituted cyclic aliphatic or aryl groups.
WO 98/16502 discloses, inter alia, ICE inhibitors of the formula: 
wherein R1 and R2 are as described in the application and the pyrrolidine ring is substituted by various groups.
While a number of caspase inhibitors have been reported, it is not clear whether they possess the appropriate pharmacological properties to be therapeutically useful. Therefore, there is a continued need for small molecule caspase inhibitors that are potent, stable, and penetrate membranes to provide effective inhibition of apoptosis in vivo. Such compounds would be extremely useful in treating the aforementioned diseases where caspase enzymes play a role.
It has now been found that compounds of this invention and pharmaceutical compositions thereof are effective as inhibitors of caspases and cellular apoptosis. These compounds have the general formula I: 
wherein:
Z is oxygen or sulfur;
R1 is hydrogen, xe2x80x94CHN2, xe2x80x94R, xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y;
R is a C1-12 aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl;
Y is an electronegative leaving group;
R2 is CO2H, CH2CO2H, or esters, amides or isosteres thereof;
R3 is a group capable of fitting into the S2 sub-site of a caspase;
R4 and R5 taken together with the intervening nitrogen form a mono-, bi- or tricyclic hetero ring system having 1-6 heteroatoms selected from nitrogen, oxygen or sulfur.
The compounds of this invention have inhibition properties across a range of caspase targets with good efficacy in cellular models of apoptosis. In addition, these compounds will have good cell penetration and pharmacokinetic properties and, as a consequence of their potency, have good efficacy against diseases where caspases are implicated.
This invention provides novel compounds, and pharmaceutically acceptable derivatives thereof, that are useful as caspase inhibitors. The invention also provides methods for using the compounds to inhibit caspase activity and to treat caspase-mediated disease states. These compounds have the general formula I: 
wherein
Z is oxygen or sulfur;
R1 is hydrogen, xe2x80x94CHN2, xe2x80x94R, xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y;
R is a C1-12 aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl;
Y is an electronegative leaving group;
R2 is CO2H, CH2CO2H, or esters, amides or isosteres thereof;
R3 is a group capable of fitting into the S2 sub-site of a caspase; and
R4 and R5 taken together with the intervening nitrogen form a mono-, bi- or tricyclic hetero ring system having 1-6 heteroatoms selected from nitrogen, oxygen or sulfur.
As used herein, the following definitions shall apply unless otherwise indicated. The term xe2x80x9caliphaticxe2x80x9d as used herein means straight chained or branched C1xe2x80x94C12 hydrocarbons which are completely saturated or which contain one or more units of unsaturation. For example, suitable aliphatic groups include substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, or alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. The term xe2x80x9calkylxe2x80x9d used alone or as part of a larger moiety refers to both straight and branched chains containing one to twelve carbon atoms. When the term alkyl is used as part of a larger moiety, as in aralkyl or heteroaralkyl, the alkyl portion will preferably contain one to six carbons. The term xe2x80x9chalogenxe2x80x9d means F, Cl, Br, or I. The term xe2x80x9carylxe2x80x9d refers to monocyclic or polycyclic aromatic ring groups having five to fourteen atoms, such as phenyl, naphthyl and anthryl. The term xe2x80x9cheterocyclic groupxe2x80x9d refers to saturated and unsaturated monocyclic or polycyclic ring systems containing one or more heteroatoms and a ring size of three to nine such as furanyl, thienyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, dioxolanyl, oxazolyl, thiazolyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyranyl, pyridinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, trithianyl, indolizinyl, indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, or phenoxazinyl. xe2x80x9cHeteroarylxe2x80x9d refers to a heterocyclic ring that is aromatic. It is understood that the compounds of this invention are limited to those that can exist in nature as stable chemical compounds.
The term xe2x80x9ccarbocyclic groupxe2x80x9d refers to saturated monocyclic or polycyclic carbon ring systems of three to fourteen carbons which may be fused to aryl or heterocyclic groups. Examples include cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, indanyl, tetrahydronaphthyl and the like.
An aliphatic, alkyl, aryl, heteroaryl, heterocyclyl, or carbocyclyl, used alone or as part of a larger moiety, refers to substituted or unsubstituted groups. When substituted, these groups may contain one or more substituents. Examples of suitable substituents include halogen, xe2x80x94R, xe2x80x94OR, xe2x80x94OH, xe2x80x94SH, xe2x80x94SR, protected OH (such as acyloxy), phenyl (Ph), substituted Ph, xe2x80x94OPh, substituted xe2x80x94OPh, xe2x80x94NO2, xe2x80x94CN, xe2x80x94NH2, xe2x80x94NHR, xe2x80x94N(R)2, xe2x80x94NHCOR, xe2x80x94NHCONHR, xe2x80x94NHCON(R)2, xe2x80x94NRCOR, xe2x80x94NHCO2R, xe2x80x94CO2R, xe2x80x94CO2H, xe2x80x94COR, xe2x80x94CONHR, xe2x80x94CON(R)2, xe2x80x94S(O)2R, xe2x80x94SONH2, xe2x80x94S(O)R, xe2x80x94SO2NHR, xe2x80x94NHS(O)2R, xe2x95x90O, xe2x95x90S, xe2x95x90NNHR, xe2x95x90NNR2, xe2x95x90Nxe2x80x94OR, xe2x95x90NNHCOR, xe2x95x90NNHCO2R, xe2x95x90NNHSO2R, or xe2x95x90NR where R is an aliphatic group or a substituted aliphatic group.
A substitutable nitrogen on a heterocyclic ring may be optionally substituted. Suitable substituents on the nitrogen include R, COR, S(O)2R, and CO2R, where R is an aliphatic group or a substituted aliphatic group.
Nitrogen and sulfur may be in their oxidized form, and nitrogen may be in a quaternized form.
The term xe2x80x9celectronegative leaving groupxe2x80x9d has the definition known to those skilled in the art (see March, Advanced Organic Chemistry, 4th Edition, John Wiley and Sons, 1992). Examples of electronegative leaving groups include halogens such as F, Cl, Br, I, aryl, and alkylsulfonyloxy groups, trifluoromethanesulfonyloxy, OR, SR, xe2x80x94OCxe2x95x90O(R), xe2x80x94OPO(R6)(R7), where R is an aliphatic group, an aryl group, an aralkyl group, a carbocyclic group, an alkyl carbocyclic group, a heterocyclic group, or an alkyl heterocyclic group; and R6 and R7 are independently selected from R or OR.
When the R2 group is in the form of an ester or amide, the present compounds undergo metabolic cleavage to the corresponding carboxylic acids, which are the active caspase inhibitors. Because they undergo metabolic cleavage, the precise nature of the ester or amide group is not critical to the working of this invention. The structure of the R2 group may range from the relatively simple diethyl amide to a steroidal ester. Examples of esters of R2 carboxylic acids include, but are not limited to, C1-12 aliphatic, such as C1-6 alkyl or C3-10 cycloalkyl, aryl, such as phenyl, aralkyl, such as benzyl or phenethyl, heterocyclyl or heterocyclylalkyl. Examples of suitable R2 heterocyclyl rings include, but are not limited to, 5-6 membered heterocyclic rings having one or two heteroatoms such as piperidinyl, piperazinyl, or morpholinyl.
Amides of R2 carboxylic acids may be primary, secondary or tertiary. Suitable substituents on the amide nitrogen include, but are not limited to, one or more groups independently selected from the aliphatic, aryl, aralkyl, heterocyclyl or heterocyclylalkyl groups described above for the R2 ester alcohol. Likewise, other prodrugs are included within the scope of this invention. See Bradley D. Anderson, xe2x80x9cProdrugs for Improved CNS Deliveryxe2x80x9d in Advanced Drug Delivery Reviews (1996), 19, 171-202.
Isosteres or bioisosteres of R2 carboxylic acids, esters and amides result from the exchange of an atom or group of atoms to create a new compound with similar biological properties to the parent carboxylic acid or ester. The bioisosteric replacement may be physicochemically or topologically based. An example of an isosteric replacement for a carboxylic acid is CONHSO2(alkyl) such as CONHSO2Me.
R3 may be any group capable of fitting into the S2 sub-site of a caspase. Such groups are known from the many caspase inhibitors that have been reported (see WO91/15577, WO93/05071, WO99/18781, WO99/47154, WO00/023421, WO9724339, EP618223, WO9816502, all of which are described above). Furthermore, the structures of several of the caspase enzymes including the S-2 subsites are also known. References to the caspase structure include the following: Blanchard H, et al., J. Mol. Biol. 302(1), 9-16 (2000); Wei Y, et al., Chem. Biol. 7(6):423-32 (2000); Lee D, et al., J Biol. Chem. 275 (21):16007-14 (2000); Blanchard H, et al., Structure Fold Des. 7(9):1125-33 (1999); Okamoto Y, et al, Chem. Pharm. Bull. (Tokyo) 47(1):11-21 (1999); Margolin N, et al, J. Biol. Chem. 272(11):7223-8 (1997); Walker N P, et al., Cell 78(2):343-52 (1994); and Wilson K P, et al., Nature 370(6487):270-5 (1994).
Whether a group will fit into the S-2 subsite will depend on the particular caspase that is being considered. The size of the subsite will range from the small S-2 subsite of caspase-3 which permits a group up to the size of a C4 aliphatic group to a relatively large subsite which permits a group having a molecular weight up to about 140 Daltons, such as a naphthyl group. The size, along with the electronic nature, of the R3 group will influence the caspase selectivity of the inhibitor. From the references provided above, one skilled in the art could readily ascertain whether a group is capable of fitting favorably into an S-2 subsite of a caspase, for example, by using standard molecular modeling programs such as Quanta or Macromodel.
R3 groups include those that are selected from hydrogen, a side chain of a natural xcex1-amino acid, or a substituted or unsubstituted group having a molecular weight up to about 140 Daltons selected from aliphatic, aryl, aralkyl, heterocyclyl, and heterocyclylalkyl groups. Examples of R3 aliphatic groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl. Examples of R3 aryl groups include phenyl, indenyl and naphthyl. Examples of R3 heterocyclic groups include pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, homopiperidinyl, and quinuclidinyl. Examples of R3 heteroaryl groups include furanyl, thienyl, pyrrolyl, oxazole, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, furazanyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophene, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, chromanyl, and isochromanyl. Each group may contain one or more substituents, as described above.
R4 and R5 taken together with the intervening nitrogen form mono-, bi- or tricyclic hetero ring system having 1-6 heteroatoms, preferably 1-4 heteroatoms. Such rings include substituted or unsubstituted indole, isoindole, indoline, indazole, purine, dihydropyridine, benzimidazole, imidazole, imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole, phenothiazine, phenoxazine, dihydrophenazine, dihydrocinnoline, dihydroquinoxaline, tetrahydroquinoline, tetrahydroisoquinoline, dihydronaphthyridine, tetrahydronaphthyridine, dihydroacridine, 5H-dibenzo[b,f]azepine, 10,11-dihydro-5H-dibenzo[b,f]azepine, xcex2-carboline, pyrido[4,3-b]indole, 2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene, thieno[3,2-b]pyrrole, or dihydrophenanthridine. Suitable substituents on R4 or R5 include one or more groups independently selected from a halogen, xe2x80x94R, xe2x80x94OR, xe2x80x94OH, xe2x80x94SH, xe2x80x94SR, protected OH (such as acyloxy), phenyl (Ph), substituted Ph, xe2x80x94OPh, substituted xe2x80x94OPh, xe2x80x94NO2, xe2x80x94CN, xe2x80x94NH2, xe2x80x94NHR, xe2x80x94N(R)2, xe2x80x94NHCOR, xe2x80x94NHCONHR, xe2x80x94NHCON(R)2, xe2x80x94NRCOR, xe2x80x94NHCO2R, xe2x80x94CO2R, xe2x80x94CO2H, xe2x80x94COR, xe2x80x94CONHR, xe2x80x94CON(R)2, xe2x80x94S(O)2R, xe2x80x94SONH2, xe2x80x94S(O)R, xe2x80x94SO2NHR, or xe2x80x94NHS(O)2R, where each R is independently selected from an aliphatic group or a substituted aliphatic group.
Compounds of this invention where R2 is COOH are gamma-ketoacids, which may exist in solution as either the open form 1 or the cyclized hemiketal form 2. The representation herein of either isomeric form is meant to include the other. Similarly, cyclization may also occur where R2 is CH2COOH, and such cyclized isomers are understood to be included when the ring open form is represented herein. 
Likewise it will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms or hydrated forms, all such forms of the compounds being within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention.
One embodiment of this invention relates to compounds that have one or more, and preferably all, of the following features:
(i) Z is oxygen.
(ii) R1 is hydrogen, xe2x80x94R, xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y. More preferably, R1 is xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y. An even more preferred R1 is xe2x80x94CH2Y. Most preferably, R1 is xe2x80x94CH2F.
(iii) R2 is CO2H or an ester, amide or isostere thereof.
(iv) R3 is a group having a molecular weight up to about 140 Daltons, such as an aliphatic or aralkyl group. More preferably, R3 is a C1xe2x80x94C4 alkyl which is a group that fits into the S2 subsite of a range of caspases.
(v) R4 and R5 taken together with the intervening nitrogen form a monocyclic, bicyclic or tricyclic heterocyclic or heteroaryl ring system wherein each ring of the system has 5-7 ring atoms.
A key feature of the present compounds is the hetero ring system formed by taking R4 and R5 together with the intervening nitrogen. Bicyclic or tricyclic heterocyclic or heteroaryl rings are preferred over monocyclic rings. Accordingly, a preferred embodiment relates to compounds having one or more, and preferably all, of the following features: (i) Z is oxygen; (ii) R1 is hydrogen, xe2x80x94R, xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y, more preferably, R1 is xe2x80x94CH2OR, xe2x80x94CH2SR, or xe2x80x94CH2Y, more preferably, R1 is xe2x80x94CH2Y, and most preferably, R1 is xe2x80x94CH2F;
(iii) R2 is CO2H or an ester, amide or isostere thereof;
(iv) R3 is a group having a molecular weight up to about 140 Daltons, such as an aliphatic or aralkyl group, more preferably a C1-4 alkyl group; and/or (v) R4 and R5 taken together with the intervening nitrogen form a bicyclic or tricyclic heterocyclic or heteroaryl ring system wherein each ring of the system has 5-7 ring atoms.
Examples of preferred monocyclic rings include triazole, piperidine, morpholine, thiomorpholine, imidazole, pyrrolidine, pyrazole, and piperazine. Examples of preferred bicyclic rings include indole, isoindole, indoline, indazole, benzimidazole, thieno[3,2-b]pyrrole, dihydroquinoxaline, dihydrocinnoline, dihydronaphthyridine, tetrahydronaphthyridine, tetrahydroquinoline, and tetrahydroisoquinoline, most preferably indole or indoline. Examples of preferred tricyclic rings include carbazole, phenothiazine, xcex2-carboline, pyrido[4,3-b]indole, 2,3,9-triazafluorene, 9-thia-2,10-diazaanthracene, 3,6,9-triazafluorene, phenoxazine, dibenzoazepine, dihydro-dibenzoazepine, dihydrophenazine, dihydroacridine, or dihydrophenanthridine, most preferably carbazole, phenothiazine or dihydrophenanthridine.
Specific examples of compounds I are shown in Table 1.
After evaluating many R4xe2x80x94Nxe2x80x94R5 heterocyclic rings, it was found that tricyclic compounds where the end rings are substantially co-planar show surprisingly superior broad caspase activity compared to acyclic analogs or other tricyclic ring systems that are not substantially co-planar. This substantial co-planarity can be achieved when the middle ring of the tricyclic ring system is a 5- or 6-membered ring, such as in a carbazole or phenothiazine ring.
Furthermore, these substantially co-planar tricyclic ring systems, as well as bicyclic ring systems such as indole and indoline, confer better broad caspase activity that the corresponding compounds where the R4xe2x80x94Nxe2x80x94R5 heterocyclic ring is monocyclic such as piperidine, piperazine or morpholine.
Accordingly, a preferred embodiment of this invention relates to compounds of formula I where R4xe2x80x94Nxe2x80x94R5 is a tricyclic ring system having 1-6 heteroatoms, preferably 1-4 heteroatoms, selected from nitrogen, oxygen or sulfur wherein the end rings of the ring system have 5-7 ring atoms and the middle ring has 5 or 6 ring atoms.
One aspect of this embodiment relates to compounds of formula II: 
where X is a bond, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94, or xe2x80x94NHxe2x80x94, and Z, R1, R2 and R3 are as described above. Where X is xe2x80x94CH2xe2x80x94, each of the methylene hydrogens may be optionally and independently replaced by xe2x80x94OR, xe2x80x94OH, xe2x80x94SR, protected OH (such as acyloxy), xe2x80x94CN, xe2x80x94NH2, xe2x80x94NHR, xe2x80x94N(R)2, xe2x80x94NHCOR, xe2x80x94NHCONHR, xe2x80x94NHCON(R)2, xe2x80x94NRCOR, xe2x80x94NHCO2R, xe2x80x94CO2R, xe2x80x94CO2H, xe2x80x94COR, xe2x80x94CONHR, xe2x80x94CON(R)2, xe2x80x94S(O)2R, xe2x80x94SONH2, xe2x80x94S(O)R, xe2x80x94SO2NHR, xe2x80x94NHS(O)2R, xe2x95x90O, xe2x95x90S, xe2x95x90NNHR, xe2x95x90NNR2, xe2x95x90Nxe2x80x94OR, xe2x95x90NNHCOR, xe2x95x90NNHCO2R, xe2x95x90NNHSO2R, or xe2x95x90NR where R is a C1-4 aliphatic group. Where X is xe2x80x94NHxe2x80x94, the NH hydrogen may be replaced by alkyl, CO(alkyl), CO2(alkyl), or SO2(alkyl). Preferred groups for R1, R2 and R3 are as described above.
The compounds of this invention may be prepared in general by methods known to those skilled in the art for analogous compounds, as illustrated by the general schemes below and by the preparative examples that follow. 
Reagents: (a) R5xe2x80x94Nxe2x95x90Cxe2x95x90Z (2); (b) NaOH/THF/H2O; (c) EDC/DMAP/HOBt; (d) i. Dess-Martin periodinane, (ii) TFA/DCM
Scheme I above shows a synthetic route for obtaining compounds where R4 is a hydrogen. Reaction of an isocyanate or thioisocyanate 2 with a lactic acid derivative 1 produces carbamate 3. The ester group of 3 is hydrolyzed using base or, when the ester is a t-butyl group, using trifluoroacetic acid to provide the acid 4, which is then coupled with the amino alcohol 5. Depending on the nature of R1 and R2 an amino ketone may be used, in place of the amino alcohol, which avoids the subsequent oxidation step. In the case of fluoromethyl ketones where R1 is CH2F, the amino alcohol 5 may be obtained according to the method of Revesz et al., Tetrahedron Lett., 1994, 35, 9693. Finally the hydroxyl group in compound 6 is oxidized and the resulting compound treated appropriately according to the nature of R2. For example, if the product I requires R to be a carboxylic acid, then R2 in 6 is preferably an ester and the final step in the scheme is a hydrolysis.
Starting isocyanates or thioisocyanates 1 are commercially available or may be made by reaction of an amine with phosgene or a phosgene equivalent (or thiophosgene for preparation of thioisocyanates) in the presence of a base such as triethylamine. The lactate derivatives are commercially available or may be made by reaction of an amino acid with a diazotization reagent such as with NaNO2. 
Reagents: (a) CDI/THF; (b) MeOTf/CH2Cl2; (c) R4R5NH (8)/THF.
Scheme II above shows a synthetic route for obtaining compounds I of this invention where R4 is an alkyl group or when R4 and R5 together form a ring. Reaction of the lactate derivative 2 with 1,1xe2x80x2-carbonyldiimidazole (CDI) gives the imidazolate 7. Methylation of 7 by methyl triflate, followed by reaction with amine 8 (see J. Med. Chem., (1996), 39, 982) provides the intermediate 3. Scheme I above shows how 3 may be converted to I. 
Reagents: (a) COCl2/CH2Cl2; (b) 1/THF.
An alternative synthetic route for obtaining compounds I of this invention where R4 is an alkyl group or when R4 and R5 together form a ring is shown in Scheme III above. Treatment of amine 8 with phosgene gives a carbamoyl chloride intermediate 9. Reaction of 9 with lactate derivative 1 provides intermediate 3. 
Reagents: (a) C13COC(O)Cl/THF; (b) R4R5NH (8), NaOH, BU4NBr.
Scheme IV above shows a synthetic route for obtaining compounds of this invention where R4 is a hydrogen or an alkylgroup or when R4 and R5 together form a ring. Reaction of hydroxy ester 1 with phosgene or a phosgene equivalent such as diphogene or triphosgene leads to chloroformate intermediate 10. Reaction of 10 with amine 8 provides intermediate 3.
The compounds of this invention are designed to inhibit caspases. Therefore, the compounds of this invention may be assayed for their ability to inhibit apoptosis, the release of IL-1xcex2 or caspase activity directly. Assays for each of the activities are described below in the Testing section and are also known in the art.
One embodiment of this invention relates to a composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
If pharmaceutically acceptable salts of the compounds of this invention are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
The compounds utilized in the compositions and methods of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
According to a preferred embodiment, the compositions of this invention are formulated for pharmaceutical administration to a mammal, preferably a human being.
Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally or intravenously.
Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
The above-described compositions are particularly useful in therapeutic applications relating to an IL-1 mediated disease, an apoptosis mediated disease, an inflammatory disease, an autoimmune disease, a destructive bone disorder, a proliferative disorder, an infectious disease, a degenerative disease, a disease associated with cell death, an excess dietary alcohol intake disease, a viral mediated disease, uveitis, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave""s disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn""s disease, psoriasis, atopic dermatitis, scarring, graft vs host disease, organ transplant rejection, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi""s sarcoma, multiple myeloma, haemorrhagic shock, sepsis, septic shock, burns, Shigellosis, Alzheimer""s disease, Parkinson""s disease, Huntington""s disease, Kennedy""s disease, prion disease, cerebral ischemia, epilepsy, myocardial ischemia, acute and chronic heart disease, myocardial infarction, congestive heart failure, atherosclerosis, coronary artery bypass graft, spinal muscular atrophy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related encephalitis, aging, alopecia, neurological damage due to stroke, ulcerative colitis, traumatic brain injury, spinal cord injury, hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, or Japanese encephalitis, various forms of liver disease, renal disease, polyaptic kidney disease, H. pylori-associated gastric and duodenal ulcer disease, HIV infection, tuberculosis, and meningitis. The compounds and compositions are also useful in treating complications associated with coronary artery bypass grafts and as a component of immunotherapy for the treatment of various forms of cancer.
The amount of compound present in the above-described compositions should be sufficient to cause a detectable decrease in the severity of the disease or in caspase activity and/or cell apoptosis, as measured by any of the assays described in the examples.
The compounds of this invention are also useful in methods for preserving cells, such as may be needed for an organ transplant or for preserving blood products. Similar uses for caspase inhibitors have been reported (Schierle et al., Nature Medicine, 1999, 5, 97). The method involves treating the cells or tissue to be preserved with a solution comprising the caspase inhibitor. The amount of caspase inhibitor needed will depend on the effectiveness of the inhibitor for the given cell type and the length of time required to preserve the cells from apoptotic cell death.
According to another embodiment, the compositions of this invention may further comprise another therapeutic agent. Such agents include, but are not limited to, thrombolytic agents such as tissue plasminogen activator and streptokinase. When a second agent is used, the second agent may be administered either as a separate dosage form or as part of a single dosage form with the compounds or compositions of this invention.
It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredients will also depend upon the particular compound and other therapeutic agent, if present, in the composition.
In a preferred embodiment, the invention provides a method of treating a mammal, having one of the aforementioned diseases, comprising the step of administering to said mammal a pharmaceutically acceptable composition described above. In this embodiment, if the patient is also administered another therapeutic agent or caspase inhibitor, it may be delivered together with the compound of this invention in a single dosage form, or, as a separate dosage form. When administered as a separate dosage form, the other caspase inhibitor or agent may be administered prior to, at the same time as, or following administration of a pharmaceutically acceptable composition comprising a compound of this invention.
In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.